Novel antibody frameworks

ABSTRACT

The present invention relates to novel antibody frameworks with advantageous properties.

FIELD OF THE INVENTION

The present invention relates to novel antibody frameworks withadvantageous properties.

BACKGROUND OF THE INVENTION

This invention relates to novel chimeric human antibody light chainframeworks, comprising framework regions I to III from Vκ and frameworkregion IV from Vλ, with advantageous properties, such as high stabilityand reduced aggregation propensity.

In the past forty years since the development of the first monoclonalantibodies (“mAbs”; Köhler & Milstein, Nature. 256 (1975) 495-7),antibodies have become an increasingly important class of biomoleculesfor research, diagnostic and therapeutic purposes. Initially, antibodieswere exclusively obtained by immunizing animals with the correspondingantigen of interest. While antibodies of non-human origin can be used inresearch and diagnostics, in therapeutic approaches the human body mayrecognize non-human antibodies as foreign and raise an immune responseagainst the non-human antibody drug substance, rendering it less or noteffective. Thus, recombinant methods have been set up to rendernon-human antibodies less immunogenic.

Initial efforts to convert non-human mAbs into less immunogenictherapeutics entailed the engineering of chimeric antibodies consistingof animal (for example rodent) variable domains and human constantregions (Boulianne et al., Nature 312, (1984) 643-646). Furtherapproaches aimed at the humanization of the rodent mAbs by introducingthe CDRs in human variable domain scaffolds (Jones et al. Nature 321(1986) 522-525; Riechmann et al., Nature 332 (1988) 323-7) or byresurfacing the variable domains (Roguska et al., Proc. Natl. Acad. Sci.USA 91 (1994) 969-973).

For the humanization by CDR loop grafting a human acceptor framework iseither chosen based on homology to the donor framework (e.g. Roguska etal., Protein Engineering 9 (1996) 895-904; WO/2008/144757 (for rabbits))or based on a preferred stability profile (Ewert et al., Methods 34(2004) 184-199). The latter concept has been utilized for thehumanization of rabbit antibodies onto a universal variable domainframework (U.S. Pat. No. 8,193,235).

With any chosen approach the resulting mAb or functional fragmentideally retains the desired pharmacodynamic properties of the donor mAb,while displaying drug-like biophysical properties and minimalimmunogenicity. With respect to the biophysical properties of mAbs orfunctional fragments thereof, the propensity for aggregation has been amajor concern for the developability of therapeutic molecules, mainlyfor the following three reasons:

First, protein aggregates generally show a higher potential to elicit animmune reaction in the host leading to the formation of anti-drugantibodies and eventually to drug neutralizing antibodies (Joubert etal., J. Biol. Chem. 287 (2012) 25266-25279).

Second, aggregates affect the manufacturing yield due to the increasedeffort for their removal (Cromwell et al., AAPS Journal 8 (2006),Article 66).

Third, off-target effects may be observed. The concern about oligomerformation is even more pronounced for applications where monovalentbinding is preferred, including bispecific (or multi-specific) antibodyformats with only one valency per target and construct, because oligomerformation in these cases results in protein conglomerates withmultivalent binding properties potentially leading to off-targeteffects. An example for such unspecific activities is the use of aconstruct with a single CD3ε-binding domain in a bispecific antibodyformat. Such a format may for example bind with one of its two bindingdomains to a cancer antigen and with its second, CD3ε-binding domainrecruiting cytotoxic T cells. Because cross-linking of the monovalentCD3ε-binding moiety is required to induce signaling through CD3ε, Tcells will only be stimulated when engaged by multiple bispecificconstructs bound to the surface of the target cell—and thereforeadopting the properties of a cross-linked molecule—resulting in aspecific T cell response that is exclusively directed towards the cancercell. On the contrary, oligomers of such a construct would exhibit theproperties of a cross-linked bispecific antibody and therefore activatecytotoxic T cells, even when not bound to cancer cells, thereby leadingto systemic activation of T cells. Such unspecific and systemicactivation of T cells could result is elevated cytokine levels leadingto adverse effects.

Furthermore, a reliable and universally applicable acceptor framework isbeneficial to enable a robust method of humanizing non-human antibodies,since cloning, expression and purification methods may be standardized.

To meet the above mentioned criteria for the humanization of non-humanmAbs the published methodology proposes the use of human consensusvariable domain framework sequences as acceptor scaffold for theengraftment of non-human complementarity determining regions. Based onthe assumption that for each amino acid position in a protein, residuesthat contribute to protein stability have been enriched in the pool ofgerm line sequences during evolution, it is the common understandingthat the closer the resulting humanized variable domains are to thehuman germ line consensus sequence of the respective variable domainfamily, the higher is the expected stability. This concept as describedby Steipe (Steipe et al., J. Mol. Biol. 1994 240 (1994) 188-92) andreviewed by Wörn (Worn et al., J. Mol. Biol. 305 (2001) 989-1010) iswidely accepted and finds wide-ranging application. Non-limitingexamples are (a) the use of consensus sequence variable domains for thehumanization of non-human antibodies (Carter et al., Proc. Natl. Acad.Sci. USA 89 (1992) 4285-4289); (b) the use of consensus sequencevariable domains to construct CDR libraries for in vitro screening ofstable target-binding antibodies (Knappik et al., J. Mol. Biol. 296(2000) 57-86); and (c) knowledge-based approaches to improve stabilityof antibody variable domains by exchanging non-consensus residues intoconsensus residues (Steipe, loc. cit.).

In addition, stabilities of the different variable domain families aredescribed with VH3 being the most stable variable heavy domain.Importantly, in case of the variable light chain domains the Vκ familyrather than the Vλ family is preferred (Ewert, loc. cit.). Inparticular, the human consensus sequences of VH3 and Vκ1 have beendescribed as having favorable biophysical properties (Ewert, loc. cit.)and as being particularly suitable for the humanization of antibodiesfrom non-human sources (use in Carter, loc. cit.).

In line with this there are several publications, in which the humanVκ1-VH3 consensus framework hu-4D5 has been used for the humanization ofrodent and rabbit antibodies (Rader, J. Biol. Chem. 275 (2000)13668-13676; WO/2005/016950; WO 2008/004834). Alternatively, a naturallyoccurring sequence belonging to the same families as hu-4D5 has beenused to generate stable humanized single-chain (scFv) fragments fromrabbit origin (U.S. Pat. No. 8,293,235; Borras et al., J. Biol. Chem.285 (2010) 9054-9066).

Importantly it has to be noted that natural selection evolved stablevariable domains always in the context of full-length antibodies, inwhich the variable domain is adjacent to and in contact with theconstant domain 1. Therefore, it may well be that certain non-consensusresidues provide better stability to the isolated variable domains, forexample in the context of the single-chain Fv (scFv) fragment. Insupport with this hypothesis, non-consensus mutations contributing tostability have been described in US patent application US 2009/0074780.

Additionally, antibody stability is of crucial importance forproduction, purification, shelf-life and, as a consequence, the cost ofgoods for antibody therapeutics. Even minor improvements in one or moreof these parameters may be highly relevant for the question of whetherresearch and development of an antibody drug are going to becommercially viable.

Thus, despite that fact that many attempts have already been made toaddress the issue of obtaining humanized antibody drug substances fromnon-human antibodies, there still remains a large unmet need to developnovel human antibody frameworks with advantageous properties, such ashigh stability and reduced aggregation propensity, wherein the humanantibody frameworks contain as few mutations as possible, ideally none,when compared to naturally occurring sequences, in order to reduce therisk of creating immunogenic sequences as far as possible. Such stablehuman frameworks could also be used to stabilize fully human antibodiesor fragments thereof for example by loop grafting or simply byexchanging the stability-contributing component between the parentantibody and the stable framework.

The solution for this problem that has been provided by the presentinvention, i.e. novel chimeric human antibody light chain frameworks,comprising framework regions I to III from Vκ and framework region IVfrom Vλ, with advantageous properties, such as high stability andreduced aggregation propensity, has so far not been achieved orsuggested by the prior art.

SUMMARY OF THE INVENTION

The present invention relates to novel chimeric human antibody lightchain frameworks, comprising framework regions I to III from Vκ andframework region IV from Vλ, with advantageous properties, such as highstability, reduced aggregation propensity and minimal immunogenicpotential.

Thus, in a first aspect, the present invention relates to an antibody VLdomain comprising (i) human Vκ framework regions I to III; (ii) CDRdomains CDR1, CDR2 and CDR3; and (iii) a framework region IV, which isselected from

-   -   a. a human Vλ germ line sequence for framework region IV,        particularly a Vλ germ line sequence selected from the list of:        SEQ ID NO. 16 to 22;    -   b. a Vλ-based sequence, which is (bi) a consensus Vλ sequence        from human Vλ germ line sequences for framework region IV,        particularly SEQ ID NO. 17; or (bii) a consensus Vλ sequence        from rearranged human Vλ sequences for framework region IV,        particularly a Vλ consensus sequence selected from the list of:        SEQ ID NO. 16 and 17; and    -   c. a Vλ-based sequence, which has one or two mutations,        particularly one mutation, compared to the closest human Vλ germ        line sequence for framework region IV;        -   provided that if, in case of b. or c, framework region IV            has the sequence FGQGTKLTVLG (SEQ ID No. 15)            -   (w) said human Vκ framework regions I to III are                different from the framework regions as found in the                list of clones: FW1.4gen (SEQ ID NO: 4), 375-FW1.4opt,                435-FW1.4opt, 509-FW1.4opt, 511-FW1.4opt, 534-FW1.4opt,                567-FW1.4opt, 578-FW1.4opt, 1-FW1.4opt, 8-FW1.4opt,                15-FW1.4opt, 19-FW1.4opt, 34-FW1.4opt, 35-FW1.4opt,                42-FW1.4opt, and 43-FW1.4opt;            -   (x) said human Vκ framework regions I to III are                different from a sequence obtainable by permutation from                the sequences of the framework regions as found in the                list of clones: FW1.4gen (SEQ ID NO: 4), 375-FW1.4opt,                435-FW1.4opt, 509-FW1.4opt, 511-FW1.4opt, 534-FW1.4opt,                567-FW1.4opt, 578-FW1.4opt, 1-FW1.4opt, 8-FW1.4opt,                15-FW1.4opt, 19-FW1.4opt, 34-FW1.4opt, 35-FW1.4opt,                42-FW1.4opt, and 43-FW1.4opt;            -   (y) said human Vκ framework regions I to III are                different from a sequence obtainable by mutation of the                sequence FW1.4gen (SEQ ID NO: 4) at one or more of                positions 15, 22, 48, 57, 74, 87, 88, 90, 92, 95, 97 and                99 (AHo numbering); or            -   (z) said human Vκ framework regions I to III comprise                not more than five mutations compared to the respective                regions in the human Vκ sequence with SEQ ID No: 8,                particularly less than five, less than four, less than                three, particularly only one or no mutation compared to                the human Vκ sequence with SEQ ID No: 8.

In a second aspect, the present invention relates to an isolatedantibody or functional fragment thereof comprising an antibody VL domainaccording to the present invention.

In a third aspect, the present invention relates to a pharmaceuticalcomposition comprising the isolated antibody or functional fragmentthereof of the present invention, and optionally a pharmaceuticallyacceptable carrier and/or excipient.

In a fourth aspect, the present invention relates to a nucleic acidsequence or a collection of nucleic acid sequences encoding the antibodyVL domain of any one of the present invention, or the isolated antibodyor functional fragment thereof of the present invention, and/or to anucleic acid sequence or nucleic acid sequences obtainable by the methodaccording to the ninth aspect of the present invention.

In a fifth aspect, the present invention relates to a vector or acollection of vectors comprising the nucleic acid sequence or acollection of nucleic acid sequences of the present invention.

In a sixth aspect, the present invention relates to a host cell,particularly an expression host cell, comprising the nucleic acidsequence or the collection of nucleic acid sequences of the presentinvention, or the vector or collection of vectors of the presentinvention.

In a seventh aspect, the present invention relates to a method forproducing the antibody VL domain of any one of the present invention, orthe isolated antibody or functional fragment thereof of the presentinvention., comprising the step of expressing the nucleic acid sequenceor the collection of nucleic acid sequences of the present invention, orthe vector or collection of vectors of the present invention, or thehost cell, particularly an expression host cell, of the presentinvention.

In an eighth aspect, the present invention relates to a method forgenerating a humanized rabbit antibody comprising the steps of:

-   -   a) immunization of rabbits with an antigen of interest;    -   b) isolating at least one antibody of interest; and    -   c) cloning of the VL CDR regions of said at least one antibody        of interest into a nucleic acid sequence encoding an antibody VL        domain according to the present invention.

In a ninth aspect, the present invention relates to a method forgenerating a nucleic acid sequence encoding an antibody VL domainaccording to the present invention, or one or more nucleic acidsequences encoding an isolated antibody or functional fragment thereofaccording to the present invention, comprising combining in one or moresteps nucleic acid sequences encoding (i) human Vκ framework regions Ito III; (ii) CDR domains CDR1, CDR2 and CDR3, and (iii) a frameworkregion IV, which is selected from

-   -   a. a human Vλ germ line sequence for framework region IV,        particularly a Vλ germ line sequence selected from the list of:        SEQ ID NO. 16 to 22;    -   b. a Vλ-based sequence, which is (bi) a consensus Vλ sequence        from human Vλ germ line sequences for framework region IV,        particularly SEQ ID NO. 17; or (bii) a consensus Vλ sequence        from rearranged human Vλ sequences for framework region IV,        particularly a Vλ consensus sequence selected from the list of:        SEQ ID NO. 16 and 17; and    -   c. a Vλ-based sequence, which has one or two mutations,        particularly one mutation, compared to the closest human Vλ germ        line sequence for framework region IV;        particularly using one of the following methods:    -   i. replacing in a nucleic acid construct, particularly in a        recombinant vector, comprising a nucleic acid sequence encoding        a human or humanized Vκ domain the Vκ framework region IV by a        framework region IV, which is selected from        -   a. a human Vλ germ line sequence for framework region IV,            particularly a Vλ germ line sequence selected from the list            of: SEQ ID NO. 16 to 22;        -   b. a Vλ-based sequence, which is (bi) a consensus Vλ            sequence from human Vλ germ line sequences for framework            region IV, particularly SEQ ID NO. 17; or (bii) a consensus            Vλ sequence from rearranged human Vλ sequences for framework            region IV, particularly a Vλ consensus sequence selected            from the list of: SEQ ID NO. 16 and 17; and        -   c. a Vλ-based sequence, which has one or two mutations,            particularly one mutation, compared to the closest human Vλ            germ line sequence for framework region IV;    -   ii. inserting in one or more steps into a nucleic acid        construct, particularly into a recombinant vector, comprising a        nucleic acid sequence encoding a framework region IV one or more        nucleic acid sequences encoding (i) human Vκ framework regions I        to III; and (ii) CDR domains CDR1, CDR2 and CDR3, wherein said        framework region IV is selected from        -   a. a human Vλ germ line sequence for framework region IV,            particularly a Vλ germ line sequence selected from the list            of: SEQ ID NO. 16 to 22;        -   b. a Vλ-based sequence, which is (bi) a consensus Vλ            sequence from human Vλ germ line sequences for framework            region IV, particularly SEQ ID NO. 17; or (bii) a consensus            Vλ sequence from rearranged human Vλ sequences for framework            region IV, particularly a Vλ consensus sequence selected            from the list of: SEQ ID NO. 16 and 17; and        -   c. a Vλ-based sequence, which has one or two mutations,            particularly one mutation, compared to the closest human Vλ            germ line sequence for framework region IV;    -   iii. mutating in a nucleic acid sequence encoding a human or        humanized Vκ domain the nucleic acid sequence encoding framework        region IV to generate a framework region IV, which is selected        from        -   a. a human Vλ germ line sequence for framework region IV,            particularly a Vλ germ line sequence selected from the list            of: SEQ ID NO. 16 to 22;        -   b. a Vλ-based sequence, which is (bi) a consensus Vλ            sequence from human Vλ germ line sequences for framework            region IV, particularly SEQ ID NO. 17; or (bii) a consensus            Vλ sequence from rearranged human Vλ sequences for framework            region IV, particularly a Vλ consensus sequence selected            from the list of: SEQ ID NO. 16 and 17; and        -   c. a Vλ-based sequence, which has one or two mutations,            particularly one mutation, compared to the closest human Vλ            germ line sequence for framework region IV; or    -   iv. replacing in one or more steps in a nucleic acid construct,        particularly in a recombinant vector, comprising a nucleic acid        sequence encoding a light chain domain comprising human Vκ        framework regions I to III, CDR domains CDR1, CDR2 and CDR3, and        a framework region IV, one or more of the nucleic acid sequences        encoding said CDR domains by nucleic acid sequence(s) encoding        the corresponding CDR domain(s) from an antibody of interest,        wherein said framework region IV is selected from        -   a. a human Vλ germ line sequence for framework region IV,            particularly a Vλ germ line sequence selected from the list            of: SEQ ID NO. 16 to 22;        -   b. a Vλ-based sequence, which is (bi) a consensus Vλ            sequence from human Vλ germ line sequences for framework            region IV, particularly SEQ ID NO. 17; or (bii) a consensus            Vλ sequence from rearranged human Vλ sequences for framework            region IV, particularly a Vλ consensus sequence selected            from the list of: SEQ ID NO. 16 and 17; and        -   c. a Vλ-based sequence, which has one or two mutations,            particularly one mutation, compared to the closest human Vλ            germ line sequence for framework region IV.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the variable domain topology outlining the framework andCDR regions.

FIG. 2 shows a sequence alignment of the variable domain sequences ofhu-4D5 (Carter, 1992) and of the tested alternative frameworks.Differences to hu-4D5 are indicated in black. For hu-4D5, only theframework regions Vκ frameworks I to IV (SEQ ID NOs: 34 to 37) and VHframeworks I to IV (SEQ ID NOs: 38 to 41) are shown.

FIG. 3 shows a comparison of the normalized time-resolved loss ofmonomer content for scFv1 to scFv10.

FIG. 4 shows a model of the interaction between framework III residueAHo101 and the lambda joining region (framework IV); VL Kappa (top; PDBID:1FVC) and VL Lambda (bottom; PDB ID 2A9M).

FIG. 5 shows an overlay of the normalized SE-HPLC chromatograms of twoscFv constructs of a humanized mouse monoclonal antibody with thenative/original variable domains (grey) and a with the VL domaincontaining a lambda framework IV (black). The normalized scFv monomerpeak is annotated with an asterisk (*), while the oligomer and aggregatepeaks are highlighted with a bracket.

DETAILED DESCRIPTION OF THE INVENTION

The peculiarity of this invention compared to former approaches forcreating human frameworks for the humanization/stabilization ofnon-human antibodies or the stabilization of human antibodies is thefact that the present inventions relates to the replacement of a κjoining segment in a κ variable light domain by a λ joining segment(framework region IV) resulting in a κ-λ chimeric variable light domainwith improved protein stability and reduced aggregation propensity. Itfurther relates to the mutation of the κ consensus residue at positionAHo101 (framework region III) and replacement by a λ consensus residueto support packing of the λ joining segment in a κ-λ chimeric variablelight domain to further improve protein stability and to further reduceaggregation propensity.

Thus, in a first aspect, the present invention relates to an antibody VLdomain comprising (i) human Vκ framework regions I to III; (ii) CDRdomains CDR1, CDR2 and CDR3; and (iii) a framework region IV, which isselected from

-   -   a. a human Vλ germ line sequence for framework region IV,        particularly a Vλ germ line sequence selected from the list of:        SEQ ID NO. 16 to 22;    -   b. a Vλ-based sequence, which is (bi) a consensus Vλ sequence        from human Vλ germ line sequences for framework region IV,        particularly SEQ ID NO. 17; or (bii) a consensus Vλ sequence        from rearranged human Vλ sequences for framework region IV,        particularly a Vλ consensus sequence selected from the list of:        SEQ ID NO. 16 and 17; and    -   c. a Vλ-based sequence, which has one or two mutations,        particularly one mutation, compared to the closest human Vλ germ        line sequence for framework region IV;        -   provided that if, in case of b. or c, framework region IV            has the sequence FGQGTKLTVLG (SEQ ID No. 15)            -   (w) said human Vκ framework regions I to III are                different from the framework regions as found in the                list of clones: FW1.4gen (SEQ ID NO: 4), 375-FW1.4opt,                435-FW1.4opt, 509-FW1.4opt, 511-FW1.4opt, 534-FW1.4opt,                567-FW1.4opt, 578-FW1.4opt, 1-FW1.4opt, 8-FW1.4opt,                15-FW1.4opt, 19-FW1.4opt, 34-FW1.4opt, 35-FW1.4opt,                42-FW1.4opt, and 43-FW1.4opt;            -   (x) said human Vκ framework regions I to III are                different from a sequence obtainable by permutation from                the sequences of the framework regions as found in the                list of clones: FW1.4gen (SEQ ID NO: 4), 375-FW1.4opt,                435-FW1.4opt, 509-FW1.4opt, 511-FW1.4opt, 534-FW1.4opt,                567-FW1.4opt, 578-FW1.4opt, 1-FW1.4opt, 8-FW1.4opt,                15-FW1.4opt, 19-FW1.4opt, 34-FW1.4opt, 35-FW1.4opt,                42-FW1.4opt, and 43-FW1.4opt;            -   (y) said human Vκ framework regions I to III are                different from a sequence obtainable by mutation of the                sequence FW1.4gen (SEQ ID NO: 4) at one or more of                positions 15, 22, 48, 57, 74, 87, 88, 90, 92, 95, 97 and                99 (AHo numbering); or            -   (z) said human Vκ framework regions I to III comprise                not more than five mutations compared to the respective                regions in the human Vκ sequence with SEQ ID No: 8,                particularly less than five, less than four, less than                three, particularly only one or no mutation compared to                the human Vκ sequence with SEQ ID No: 8.

In the context of the present invention, the clones 375-FW1.4opt,435-FW1.4opt, 509-FW1.4opt, 511-FW1.4opt, 534-FW1.4opt, 567-FW1.4opt,578-FW1.4opt, 1-FW1.4opt, 8-FW1.4opt, 15-FW1.4opt, 19-FW1.4opt,34-FW1.4opt, 35-FW1.4opt, 42-FW1.4opt, and 43-FW1.4opt refer to theclones listed in Borras et al. (loc. cit.). These clones are variants ofthe VL domain FW1.4gen (SEQ ID NO: 4), which differ in certain positionsin the VL framework regions from those of FW1.4gen (SEQ ID NO: 4) asshown in Table 5.

In a particular embodiment, said framework region IV is not FGQGTKLTVLG(SEQ ID No. 15).

In the context of the present invention, the term “antibody” is used asa synonym for “immunoglobulin” (Ig), which is defined as a proteinbelonging to the class IgG, IgM, IgB, IgA, or IgD (or any subclassthereof), and includes all conventionally known antibodies andfunctional fragments thereof. A “functional fragment” of anantibody/immunoglobulin is defined as a fragment of anantibody/immunoglobulin (e.g., a variable region of an IgG) that retainsthe antigen-binding region. An “antigen-binding region” of an antibodytypically is found in one or more hypervariable region(s) of anantibody, i.e., the CDR-1, -2, and/or -3 regions; however, the variable“framework” regions can also play an important role in antigen binding,such as by providing a scaffold for the CDRs.

In the context of the present invention, the numbering system suggestedby Honegger & Pluckthun is used (Honegger & Pluckthun, J. Mol. Biol. 309(2001) 657-670), unless specifically mentioned otherwise, Furthermore,the following residues are defined as CDR regions: CDR-L1: L24-L42;CDR-L2: L58-L72; CDR-L3: L107-L138; CDR-H1: H27-H42; CDR-H2: H57-H76;CDR-H3: H109-H138. Preferably, the “antigen-binding region” comprises atleast amino acid residues 4 to 149 of the variable light (VL) chain and5 to 144 of the variable heavy (VH) chain, more preferably amino acidresidues 3 to 149 of VL and 4 to 146 of VH, and particularly preferredare the complete VL and VH chains (amino acid positions 1 to 149 of VLand 1 to 149 of VH; numbering according to FIG. 2). The framework andCDR regions are indicated in FIG. 2. A preferred class ofimmunoglobulins for use in the present invention is IgG. “Functionalfragments” of the invention include the domain of a F(ab′)₂ fragment, aFab fragment and scFv. The F(ab′)₂ or Fab may be engineered to minimizeor completely remove the intermolecular disulphide interactions thatoccur between the CH1 and CL domains. The antibodies or functionalfragments thereof of the present invention may be part of bi- ormultifunctional constructs, as further described in Sections [0055] to[0058].

In the context of the present invention the terms “Vκ” and “Vλ” refer tofamilies of antibody light chain sequences that are grouped according tosequence identity and homology. Methods for the determination ofsequence homologies, for example by using a homology search matrix suchas BLOSUM (Henikoff, S. & Henikoff, J. G., Proc. Natl. Acad. Sci. USA 89(1992) 10915-10919), and methods for the grouping of sequences accordingto homologies are well known to one of ordinary skill in the art. Forboth Vκ and Vλ different subfamilies can be identified (see, forexample, Knappik, loc. cit., which groups Vκ in Vκ1 to Vκ4 and Vλ in Vλ1to Vλ3).

In the context of the present invention, the term “a sequence obtainableby permutation from the sequences of the framework regions as found inthe list of clones: . . . ” refers to sequences that can be created byusing (i) either the amino acid residue present at a given position inall sequences comprised in said list (see Borras, loc. cit.) or (ii) forthe positions that have been optimized in Borras (Borras, loc. cit.),any one of the amino acid residues present at one of the diversifiedposition in said sequences (positions 15, 22, 40, 49, 58, 69, 70, 72,74, 77, 79, and 81 in Borras (Borras, loc. cit.); corresponding to AHopositions 15, 22, 48, 57, 74, 87, 88, 90, 92, 95, 97 and 99).

In one embodiment of the present invention, the amino acid residue inposition AHo101 (position 101 according to the numbering system ofHonegger and Plückthun) in framework region III is an amino acid residuepresent at that position in a human Vλ consensus sequence, particularlywherein said amino acid residue is different from phenylalanine, moreparticularly wherein said amino acid residue is glutamic acid.

In one embodiment of the present invention, said Vκ framework regions Ito III belong to a Vκ domain subfamily selected from Vκ1, Vκ2, Vκ3, andVκ4, particularly to the Vκ1 family.

In the context of the present invention, the Vκ domain subfamilies arerepresented by the consensus sequences shown in SEQ ID NOs: 23 to 26. Agiven antibody variable light chain domain is regarded as belonging to aVκ domain subfamily, if it shows the highest degree of sequence homologywith said Vκ domain subfamily, when using the methods listed in Section[0039].

In particular embodiments, the Vκ framework regions I to III comprisenot more than five mutations compared to (a) the closest human germ linesequence, or (b) one of the consensus sequences with SEQ ID NOs: 23 to26, particularly SEQ ID NO: 23; particularly less than five, less thanfour, less than three, particularly only one or no mutation compared to(a) the closest human germ line sequence, or (b) one of the consensussequences with SEQ ID NOs: 23 to 26, particularly SEQ ID NO: 23.

In one embodiment of the present invention, said Vκ framework regions Ito III are the framework regions present in a sequence selected from SEQID NO: 2 and SEQ ID NO: 8, particularly SEQ ID NO: 8.

In one embodiment of the present invention, said CDR domains CDR1, CDR2and CDR3 are independently selected from (i) CDR domains CDR1, CDR2 andCDR3 from a parental non-human antibody with specificity for an antigenof interest, particularly from a parental rabbit antibody or from aparental rodent antibody, particularly a parental mouse or rat antibody;(ii) CDR domains CDR1, CDR2 and CDR3 from a parental human or humanizedantibody comprising a Vκ domain, particularly from an antibody approvedfor therapy or otherwise being commercialized; (iii) CDR domains CDR1,CDR2 and CDR3 derived from the CDR domains according to (i) or (ii),particularly CDR domains obtained by optimizing one or more of the CDRdomains according to (i) or (ii); and (iv) a CDR domain to be replacedby one or more CDR domains according to (i), (ii) and/or (iii).

In one embodiment of the present invention, the framework regions I toIV are a combination of framework regions as found in a sequenceselected from: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12,and SEQ ID NO: 13.

In a second aspect, the present invention relates to an isolatedantibody or functional fragment thereof comprising an antibody VL domainaccording to the present invention.

In particular embodiments, the antibody VL domain has bindingspecificity for a target of interest.

As used herein, a binding molecule is “specific to/for”, “specificallyrecognizes”, or “specifically binds to” a target, such as for examplehuman CD3, when such binding molecule is able to discriminate betweensuch target biomolecule and one or more reference molecule(s), sincebinding specificity is not an absolute, but a relative property. In itsmost general form (and when no defined reference is mentioned),“specific binding” is referring to the ability of the binding moleculeto discriminate between the target biomolecule of interest and anunrelated biomolecule, as determined, for example, in accordance with aspecificity assay methods known in the art. Such methods comprise, butare not limited to Western blots, ELISA, RIA, ECL, IRMA tests andpeptide scans. For example, a standard ELISA assay can be carried out.The scoring may be carried out by standard colour development (e.g.secondary antibody with horseradish peroxide and tetramethyl benzidinewith hydrogen peroxide). The reaction in certain wells is scored by theoptical density, for example, at 450 nm. Typical background (=negativereaction) may be about 0.1 OD; typical positive reaction may be about 1OD. This means the ratio between a positive and a negative score can be10-fold or higher. Typically, determination of binding specificity isperformed by using not a single reference biomolecule, but a set ofabout three to five unrelated biomolecules, such as milk powder, BSA,transferrin or the like.

In the context of the present invention, the term “about” or“approximately” means between 90% and 110% of a given value or range.

However, “specific binding” also may refer to the ability of a bindingmolecule to discriminate between the target biomolecule and one or moreclosely related biomolecule(s), which are used as reference points.Additionally, “specific binding” may relate to the ability of a bindingmolecule to discriminate between different parts of its target antigen,e.g. different domains, regions or epitopes of the target biomolecule,or between one or more key amino acid residues or stretches of aminoacid residues of the target biomolecule.

In the context of the present invention, the term “epitope” refers tothat part of a given target biomolecule that is required for specificbinding between the target biomolecule and a binding molecule. Anepitope may be continuous, i.e. formed by adjacent structural elementspresent in the target biomolecule, or discontinuous, i.e. formed bystructural elements that are at different positions in the primarysequence of the target biomolecule, such as in the amino acid sequenceof a protein as target, but in close proximity in the three-dimensionalstructure, which the target biomolecule adopts, such as in the bodilyfluid.

In one embodiment of the present invention, the isolated antibody orfunctional fragment thereof is selected from: an IgG antibody, a Fab andan scFv fragment.

In another particular embodiment of the present invention, the isolatedantibody or functional fragment thereof is a bispecific construct whichis an antibody format selected from the group consisting of asingle-chain diabody (scDb), a tandem scDb (Tandab), a linear dimericscDb (LD-scDb), a circular dimeric scDb (CD-scDb), a bispecific T-cellengager (BiTE; tandem di-scFv), a tandem tri-scFv, a tri(a)body,bispecific Fab2, di-miniantibody, tetrabody, scFv-Fc-scFv fusion,di-diabody, DVD-Ig, IgG-scFab, scFab-dsscFv, Fv2-Fc, IgG-scFv fusions,such as bsAb (scFv linked to C-terminus of light chain), Bs1Ab (scFvlinked to N-terminus of light chain), Bs2Ab (scFv linked to N-terminusof heavy chain), Bs3Ab (scFv linked to C-terminus of heavy chain), Ts1Ab(scFv linked to N-terminus of both heavy chain and light chain), Ts2Ab(dsscFv linked to C-terminus of heavy chain), and Knob-into-Holes (KiHs)(bispecific IgGs prepared by the KiH technology) and DuoBodies(bispecific IgGs prepared by the Duobody technology). Particularlysuitable for use herein is a single-chain diabody (scDb), in particulara bispecific monomeric scDb.

The bispecific scDb, in particular the bispecific monomeric scDb,particularly comprises two variable heavy chain domains (VH) orfragments thereof and two variable light chain domains (VL) or fragmentsthereof connected by linkers L1, L2 and L3 in the orderVHA-L1-VLB-L2-VHB-L3-VLA, VHA-L1-VHB-L2-VLB-L3-VLA,VLA-L1-VLB-L2-VHB-L3-VHA, VLA-L1-VHB-L2-VLB-L3-VHA,VHB-L1-VLA-L2-VHA-L3-VLB, VHB-L1-VHA-L2-VLA-L3-VLB,VLB-L1-VLA-L2-VHA-L3-VHB or VLB-L1-VHA-L2-VLA-L3-VHB, wherein the VLAand VHA domains jointly form the antigen binding site for the firstantigen, and VLB and VHB jointly form the antigen binding site for thesecond antigen.

The linker L1 particularly is a peptide of 2-10 amino acids, moreparticularly 3-7 amino acids, and most particularly 5 amino acids, andlinker L3 particularly is a peptide of 1-10 amino acids, moreparticularly 2-7 amino acids, and most particularly 5 amino acids. Themiddle linker L2 particularly is a peptide of 10-40 amino acids, moreparticularly 15-30 amino acids, and most particularly 20-25 amino acids.

The bispecific constructs of the present invention can be produced usingany convenient antibody manufacturing method known in the art (see,e.g., Fischer, N. & Leger, O., Pathobiology 74 (2007) 3-14 with regardto the production of bispecific constructs; Hornig, N. & Färber-Schwarz,A., Methods Mol. Biol. 907 (2012)713-727, and WO 99/57150 with regard tobispecific diabodies and tandem scFvs). Specific examples of suitablemethods for the preparation of the bispecific construct of the presentinvention further include, inter alia, the Genmab (see Labrijn et al.,Proc. Natl. Acad. Sci. USA 110 (2013) 5145-5150) and Merus (see de Kruifet al., Biotechnol. Bioeng. 106 (2010) 741-750) technologies. Methodsfor production of bispecific antibodies comprising a functional antibodyFc part are also known in the art (see, e.g., Zhu et al., Cancer Lett.86 (1994) 127-134); and Suresh et al., Methods Enzymol. 121 (1986)210-228).

These methods typically involve the generation of monoclonal antibodies,for example by means of fusing myeloma cells with the spleen cells froma mouse that has been immunized with the desired antigen using thehybridoma technology (see, e.g., Yokoyama et al., Curr. Protoc. Immunol.Chapter 2, Unit 2.5, 2006) or by means of recombinant antibodyengineering (repertoire cloning or phage display/yeast display) (see,e.g., Chames & Baty, FEMS Microbiol. Letters 189 (2000) 1-8), and thecombination of the antigen-binding domains or fragments or parts thereofof two different monoclonal antibodies to give a bispecific constructusing known molecular cloning techniques.

In one embodiment of the present invention, the isolated antibody orfunctional fragment thereof comprises a VH domain belonging to a VHdomain subfamily selected from VH1A, VH1B, VH2, VH3, VH4, VH5, and VH6,particularly to a VH domain subfamily VH3 or VH4, particularly to the VHdomain subfamily VH3.

In the context of the present invention, the VH domain subfamilies arerepresented by the consensus sequences shown in SEQ ID NOs: 27 to 33. Agiven antibody variable heavy chain domain is regarded as belonging to aVH domain subfamily, if it shows the highest degree of sequence homologywith said VH domain subfamily, when using the methods listed in Section[0039].

In particular embodiments, the VH domain comprises not more than fivemutations compared to (a) the closest human germ line sequence, or (b)one of the consensus sequences with SEQ ID NOs: 28 to 34, particularlySEQ ID NOs: 30; particularly less than five, less than four, less thanthree, particularly only one or no mutation compared to (a) the closesthuman germ line sequence, or (b) one of the consensus sequences with SEQID NOs: 27 to 33, particularly SEQ ID NOs: 30.

In a third aspect, the present invention relates to a pharmaceuticalcomposition comprising the isolated antibody or functional fragmentthereof of the present invention, and optionally a pharmaceuticallyacceptable carrier and/or excipient.

In a fourth aspect, the present invention relates to a nucleic acidsequence or a collection of nucleic acid sequences encoding the antibodyVL domain of any one of the present invention, or the isolated antibodyor functional fragment thereof of the present invention, and/or to anucleic acid sequence or nucleic acid sequences obtainable by the methodaccording to the ninth aspect of the present invention.

In a fifth aspect, the present invention relates to a vector or acollection of vectors comprising the nucleic acid sequence or acollection of nucleic acid sequences of the present invention.

In a sixth aspect, the present invention relates to a host cell,particularly an expression host cell, comprising the nucleic acidsequence or the collection of nucleic acid sequences of the presentinvention, or the vector or collection of vectors of the presentinvention.

In a seventh aspect, the present invention relates to a method forproducing the antibody VL domain of any one of the present invention, orthe isolated antibody or functional fragment thereof of the presentinvention., comprising the step of expressing the nucleic acid sequenceor the collection of nucleic acid sequences of the present invention, orthe vector or collection of vectors of the present invention, or thehost cell, particularly an expression host cell, of the presentinvention.

In an eighth aspect, the present invention relates to a method forgenerating a humanized rabbit antibody or rodent antibody, particularlya mouse or rat antibody, comprising the steps of:

-   -   a) immunization of rabbits or rodents, particularly mice or        rats, with an antigen of interest;    -   b) isolating at least one antibody of interest; and    -   c) cloning of the VL CDR regions of said at least one antibody        of interest into a nucleic acid sequence encoding an antibody VL        domain according to the present invention.

In a particular embodiment, the framework region IV in said antibody VLdomain is not FGQGTKLTVLG (SEQ ID No. 15), when generating a humanizedrabbit antibody.

In particular embodiments, the method of the present invention furthercomprises one or more of the steps of:

-   -   aa. clonal isolation of affinity matured memory B-cells that        interact with the antigen of interest, particularly by using        fluorescence activated cell-sorting;    -   ab. cultivation of single B cells in a co-cultivation system        that does not require immortalization of single B cell clones;    -   ac. screening of B cell culture supernatants in a cell-based        ELISA to identify at least one antibody binding to the antigen        of interest; and/or    -   ad. cloning of the VH CDR regions of at least one antibody into        a nucleic acid sequence encoding a human antibody VH domain.

Methods for the humanization of rabbit antibodies or rodent antibodiesare well known to anyone of ordinary skill in the art (see, for example,Borras, loc. cit.; Rader et al, The FASEB Journal, express article10.1096/fj.02-0281fje, published online Oct. 18, 2002; Yu et al (2010) AHumanized Anti-VEGF Rabbit Monoclonal Antibody Inhibits Angiogenesis andBlocks Tumor Growth in Xenograft Models. PLoS ONE 5(2): e9072.doi:10.1371/journal.pone.0009072). The immunization of the rabbits orrodents may be performed with the antigen of interest as such, such as aprotein, or, in the case of peptide or protein antigens, by DNAimmunization of a rabbit with a nucleic acid, e.g. a plasmid, encodingthe peptides or proteins of interest.

In a ninth aspect, the present invention relates to a method forgenerating a nucleic acid sequence encoding an antibody VL domainaccording to the present invention, or one or more nucleic acidsequences encoding an isolated antibody or functional fragment thereofaccording to the present invention, comprising combining in one or moresteps nucleic acid sequences encoding (i) human Vκ framework regions Ito III; (ii) CDR domains CDR1, CDR2 and CDR3, and (iii) a frameworkregion IV, which is selected from

-   -   a. a human Vλ germ line sequence for framework region IV,        particularly a Vλ germ line sequence selected from the list of:        SEQ ID NO. 16 to 22;    -   b. a Vλ-based sequence, which is (bi) a consensus Vλ sequence        from human Vλ germ line sequences for framework region IV,        particularly SEQ ID NO. 17; or (bii) a consensus Vλ sequence        from rearranged human Vλ sequences for framework region IV,        particularly a Vλ consensus sequence selected from the list of:        SEQ ID NO. 16 and 17; and    -   c. a Vλ-based sequence, which has one or two mutations,        particularly one mutation, compared to the closest human Vλ germ        line sequence for framework region IV;        particularly using one of the following methods:    -   i. replacing in a nucleic acid construct, particularly in a        recombinant vector, comprising a nucleic acid sequence encoding        a human or humanized Vκ domain the Vκ framework region IV by a        framework region IV, which is selected from        -   a. a human Vλ germ line sequence for framework region IV,            particularly a Vλ germ line sequence selected from the list            of: SEQ ID NO. 16 to 22;        -   b. a Vλ-based sequence, which is (bi) a consensus Vλ            sequence from human Vλ germ line sequences for framework            region IV, particularly SEQ ID NO. 17; or (bii) a consensus            Vλ sequence from rearranged human Vλ sequences for framework            region IV, particularly a Vλ consensus sequence selected            from the list of: SEQ ID NO. 16 and 17; and        -   c. a Vλ-based sequence, which has one or two mutations,            particularly one mutation, compared to the closest human Vλ            germ line sequence for framework region IV;    -   ii. inserting in one or more steps into a nucleic acid        construct, particularly into a recombinant vector, comprising a        nucleic acid sequence encoding a framework region IV one or more        nucleic acid sequences encoding (i) human Vκ framework regions I        to III; and (ii) CDR domains CDR1, CDR2 and CDR3, wherein said        framework region IV is selected from        -   a. a human Vλ germ line sequence for framework region IV,            particularly a Vλ germ line sequence selected from the list            of: SEQ ID NO. 16 to 22;        -   b. a Vλ-based sequence, which is (bi) a consensus Vλ            sequence from human Vλ germ line sequences for framework            region IV, particularly SEQ ID NO. 17; or (bii) a consensus            Vλ sequence from rearranged human Vλ sequences for framework            region IV, particularly a Vλ consensus sequence selected            from the list of: SEQ ID NO. 16 and 17; and        -   c. a Vλ-based sequence, which has one or two mutations,            particularly one mutation, compared to the closest human Vλ            germ line sequence for framework region IV;    -   iii. mutating in a nucleic acid sequence encoding a human or        humanized Vκ domain the nucleic acid sequence encoding framework        region IV to generate a framework region IV, which is selected        from        -   a. a human Vλ germ line sequence for framework region IV,            particularly a Vλ germ line sequence selected from the list            of: SEQ ID NO. 16 to 22;        -   b. a Vλ-based sequence, which is (bi) a consensus Vλ            sequence from human Vλ germ line sequences for framework            region IV, particularly SEQ ID NO. 17; or (bii) a consensus            Vλ sequence from rearranged human Vλ sequences for framework            region IV, particularly a Vλ consensus sequence selected            from the list of: SEQ ID NO. 16 and 17; and        -   c. a Vλ-based sequence, which has one or two mutations,            particularly one mutation, compared to the closest human Vλ            germ line sequence for framework region IV; or    -   iv. replacing in one or more steps in a nucleic acid construct,        particularly in a recombinant vector, comprising a nucleic acid        sequence encoding a light chain domain comprising human Vκ        framework regions I to III, CDR domains CDR1, CDR2 and CDR3, and        a framework region IV, one or more of the nucleic acid sequences        encoding said CDR domains by nucleic acid sequence(s) encoding        the corresponding CDR domain(s) from an antibody of interest,        wherein said framework region IV is selected from    -   a. a human Vλ germ line sequence for framework region IV,        particularly a Vλ germ line sequence selected from the list of:        SEQ ID NO. 16 to 22;    -   b. a Vλ-based sequence, which is (bi) a consensus Vλ sequence        from human Vλ germ line sequences for framework region IV,        particularly SEQ ID NO. 17; or (bii) a consensus Vλ sequence        from rearranged human Vλ sequences for framework region IV,        particularly a Vλ consensus sequence selected from the list of:        SEQ ID NO. 16 and 17; and    -   c. a Vλ-based sequence, which has one or two mutations,        particularly one mutation, compared to the closest human Vλ germ        line sequence for framework region IV;

In particular embodiments of the methods of the present invention,wherein in the case of (b) or (c) framework region IV has the sequenceFGQGTKLTVLG (SEQ ID No. 15)

-   -   (w) said human Vκ framework regions I to III are different from        the framework regions as found in the list of clones: FW1.4gen        (SEQ ID NO: 4), 375-FW1.4opt, 435-FW1.4opt, 509-FW1.4opt,        511-FW1.4opt, 534-FW1.4opt, 567-FW1.4opt, 578-FW1.4opt,        1-FW1.4opt, 8-FW1.4opt, 15-FW1.4opt, 19-FW1.4opt, 34-FW1.4opt,        35-FW1.4opt, 42-FW1.4opt, and 43-FW1.4opt;    -   (x) said human Vκ framework regions I to III are different from        a sequence obtainable by permutation from the sequences of the        framework regions as found in the list of clones: FW1.4gen (SEQ        ID NO: 4), 375-FW1.4opt, 435-FW1.4opt, 509-FW1.4opt,        511-FW1.4opt, 534-FW1.4opt, 567-FW1.4opt, 578-FW1.4opt,        1-FW1.4opt, 8-FW1.4opt, 15-FW1.4opt, 19-FW1.4opt, 34-FW1.4opt,        35-FW1.4opt, 42-FW1.4opt, and 43-FW1.4opt;    -   (y) said human Vκ framework regions I to III are different from        a sequence obtainable by mutation of the sequence FW1.4gen (SEQ        ID NO: 4) at one or more of positions 15, 22, 48, 57, 74, 87,        88, 90, 92, 95, 97 and 99 (AHo numbering); or    -   (z) said human Vκ framework regions I to III comprise not more        than five mutations compared to the respective regions in the        human Vκ sequence with SEQ ID No: 8, particularly less than        five, less than four, less than three, particularly only one or        no mutation compared to the human Vκ sequence with SEQ ID No: 8.

In a particular embodiment, said framework region IV is not FGQGTKLTVLG(SEQ ID No. 15), when one or more of said CDR domain(s) are of rabbitorigin.

EXAMPLES

The following examples illustrate the invention without limiting itsscope.

Example 1 Construction of scFv Constructs with Exemplary Rabbit CDRs

It was our aim to identify variable domains that show improved stabilitywith respect to unfolding and aggregation tendency. In addition, thesedomains should be as close as possible to the human germ line repertoireto minimize the risk eliciting an immune response in human beings.Surprisingly we found that the combination of a VH3 consensus frameworkwith a chimeric VL domain composed of framework regions I to III from aconsensus Vκ1 and the framework region IV (see FIG. 1) from different Vλdomains resulted in a scFv construct of superior biophysical properties.

Starting point for our invention was the human VH3 and Vκ1 consensusframework Hu-4D5 as it was published already in 1992 (Carter, loc.cit.). We engrafted the CDRs of an exemplary rabbit anti-TNFα antibody(W0120091155723) onto the hu-4D5 variable domains as described (Rader2000, loc. cit.; WO 2005/016950; WO 2008/004834). The humanized variabledomains were linked by a flexible peptide linker as described by Borras,loc. cit, resulting in a single-chain Fv (scFv) fragment (scFv1).

In addition to hu-4D5, another published framework solution was tested,namely the framework FW1.4gen (scFv2), of which extensive biophysicaldata is published (Borras, loc. cit.). The framework FW1.4gen includesseveral amino acid substitutions when compared to hu-4D5 thus deviatingfrom the human consensus. In the VH, such differences probably resultfrom affinity maturation, on one hand because it originates from a humancDNA library, containing sequences of mature antibodies that haveundergone somatic hypermutation, rather than germline sequences, and onthe other hand because several mutations have been introduced by theauthors for purpose of accommodating rabbit CDRs. In the VL no or onlyfew mutations have been deliberately introduced, suggesting that mostdifferences result from the germ line sequence used somatichypermutation or possibly are artifacts resulting from library cloningprocedures.

Experimental determination of the thermal unfolding of the twoconstructs based either on hu-4D5 or FW1.4gen (scFv1 and scFv2,respectively) showed a superior performance of scFv1, the framework withhigher homology to the human consensus sequence, thus being in line withthe literature. Surprisingly this superior stability did not convertinto a higher stability with respect to monodispersity during storageunder stress conditions (see Table 4). On the contrary, scFv2 showedbetter stability here.

In an attempt to rationalize the observed differences in stability thesequence variation in the frameworks were more closely examined. Astretch of five amino acids in the framework IV region was identifiedthat led to impaired stability of the monomeric state in the stressstability study when converted to the consensus sequence of Vκ1 inFW1.4gen (scFv3) (see (FIG. 3).

This finding was highly unexpected since it contradicts the commonunderstanding that the consensus sequence, in this case of Vκ1, wouldpresent the most favored solution with regard to the stability of therespective variable domain. Based on this discovery we set out tofurther examine the determinants of domain stability in the modelsystem. As the five amino acids in framework region IV of FW1.4 made thejoining segment (framework region IV) resembling a λ-type frameworkregion IV sequence rather than a K-type, we hypothesized that λ-typejoining segments may be favorable over κ-type joining segments for thestability of the Vκ1-VH3 variable domains.

Indeed we found that the full replacement of Vκ1 framework region IV bythe corresponding Vλ framework region (scFv5, scFv9) led to favorablestability profiles compared to scFv1 or scFv2 in terms of both themidpoint of thermal unfolding (Table 3) and the stability of themonomeric state during the stress stability study (Table 4).Furthermore, the introduction of different truncated Vλ framework regionIV germline motifs into the background of the Vκ1 consensus framework(scFv7, scFv10) showed some stabilizing effects. Importantly, fullreplacement of Vκ1 framework region IV by a Vλ region IV has a lowerpotential to be immunogenic as all individual framework regions remainidentical to germline or to germline consensus with this approach.

In addition, position AHo101 in framework III was identified bycomparing structural models of Vκ and Vλ variable domains to becontributing to the packing of framework region IV (FIG. 4). Theintroduction of a Vλ consensus residue at this position (scFv4, scFv6,scFv8) led to a further increase of the domain stability in thermalunfolding (Table 3) and stress stability (Table 4).

In summary, it has been found that in the context of scFv variabledomain constructs, a superior stability profile is achieved by combiningconsensus framework regions IV from different Vλ germline genes with theframework regions I to III of a Vκ sequence. The stability of theseartificial and chimeric variable domains is further improved by themodification F101E (according to the AHo numbering scheme) in frameworkregion III of the variable light domain, a position that is in closespatial proximity to framework region IV.

Importantly, based on published cases (Schäfer, Protein Engineering,Design & Selection 25 (2012) 485-505) it is expected that the preferredproperties of the variable domains translate into other antibody formatsas well.

An exemplary rabbit binder was identified from the literature (WO2009/155723) and its CDRs were grafted onto the respective variabledomains of a human consensus VH3/Vκ1 framework (Rader 2000, loc. cit.;WO/2005/016950; WO 2008/004834; U.S. Pat. No. 8,293,235), using the CDRdefinitions as published in Borras et al (Borras, loc. cit.). For theloop grafting of the rabbit CDRs the sequence stretches CDR-L1(L24-L42), CDR-L2 (L58-L72), CDR-L3 (L107-L138), CDR-H1 (H27-H42),CDR-H2 (H57-H76), CDR-H3 (H109-H138) according to the numbering byHonegger (Honegger & Plückthun, loc. cit.; see FIG. 2) were transferredonto the human frameworks. Based on these humanized variable domains thescFv constructs were generated by connecting the VL and VH by a flexibleGly4-Ser linker, thus resulting in a configuration ofNH₂-VL-linker-VH-COOH.

Methods

Construct Design and Manufacture

The resulting amino acid sequence was de novo synthesized and clonedinto an adapted expression vector for E. coli expression that is basedon a pET26b(+) backbone (Novagen). The expression construct wastransformed into the E. coli strain BL12 (DE3) (Novagen) and the cellswere cultivated in 2YT medium (Sambrook, J., et al., Molecular Cloning:A Laboratory Manual) as a starting culture. Expression cultures wereinoculated and incubated in baffled flasks at 37° C. and 200 rpm. Oncethe OD600 nm of 1 was reached protein expression was induced by theaddition of IPTG at a final concentration of 0.5 mM. After overnightexpression the cells were harvested by centrifugation at 4000 g. For thepreparation of inclusion bodies the cell pellet was resuspended in IBResuspension Buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM EDTA, 0.5%Triton X-100). The cell slurry was supplemented with 1 mM DTT, 0.1 mg/mLLysozyme, 10 mM Leupeptin, 100 μM PMSF and 1 μM Pepstatin. Cells werelysed by 3 cycles of ultrasonic homogenization while being cooled onice. Subsequently 0.01 mg/mL DNAse was added and the homogenate wasincubated at room temperature for 20 min. The inclusion bodies weresedimented by centrifugation at 10,000 g and 4° C. The IBs wereresuspended in IB Resuspension Buffer and homogenized by sonicationbefore another centrifugation. In total a minimum of 3 washing stepswith IB Resuspension Buffer were performed and subsequently 2 washeswith IB Wash Buffer were performed to yield the final IBs.

For protein refolding the isolated IBs were resuspended inSolubilization Buffer (100 mM Tris/HCl pH 8.0, 6 M Gdn-HCl, 2 mM EDTA)in a ratio of 5 mL per g of wet IBs. The solubilization was incubatedfor 30 min at room temperature until DTT was added at a finalconcentration of 20 mM and the incubation was continued for another 30min. After the solubilization is completed the solution is cleared by 10min centrifugation at 8500 g and 4° C. The refolding is performed byrapid dilution at a final protein concentration of 0.5 g/L of thesolubilized protein in Refolding Buffer (typically: 100 mM Tris-HCl pH8.0, 4.0 M Urea, 5 mM Cysteine,1 mM Cystine). The refolding reaction isroutinely incubated for a minimum of 14 h. The resulting proteinsolution is concentrated and buffer exchanged by diafiltration intoNative Buffer (50 mM Citrate-Phosphate pH 6.4, 200 mM NaCl). Therefolded protein is purified by size-exclusion chromatography on asuitable resin material (e.g. Superdex 75, GE Healthcare). The isolatedmonomer fraction is analyzed by size-exclusion HPLC, SDS-PAGE for purityand UV/Vis spectroscopy for protein content. The protein concentrationis adjusted to the required levels and the stability analysis isperformed.

Comparison of Structural Models

The three-dimensional structures of variable domains VL Kappa and VLLambda were compared using the example of structural models available inthe PDB (PDB ID 1FVC and PDB ID 2A9M, respectively). The analysis of thepacking of the VL framework region IV revealed differences in theside-chain orientation from position 147 onwards. In addition theorientation of the amino acid side chain at position 101 differed in theVκ and Vλ structures. As illustrated by FIG. 4 the different packing ofthe central amino acid (position 101) is apparent. In the case of the Vκthe phenylalanine 101 points in the core of the domain, in the lambdavariable domain, however, the glutamate at position 101 is solventexposed and the V147 (arrow) is in turn positioned into the hydrophobiccore. Based on this observation variable domains containing the aminoacid exchange F101E were generated (SEQ ID No: 8, 11 and 13) toaccommodate the Vλ specific packing of the Vλ framework IV.

Example 2 Determination of Biophysical Data for scFv Constructs

Thermal Unfolding

The midpoint of transition for the thermal unfolding of the testedconstructs was determined by Differential Scanning Fluorimetry (DSF),essentially as described by Niesen (Niesen et al., Nat Protoc. 2 (2007)2212-21). The DSF assay is performed in a qPCR machine (e.g. MX3005p,Agilent Technologies). The samples were diluted in buffer(citrate-phosphate pH 6.4, 0.25 M NaCl) containing a final concentrationof 5× SYPRO orange in a total volume of 25 μL. In a buffer scoutingexperiment the pH dependence of the unfolding temperature was determinedand comparable pH characteristics was observed for all constructs.Samples were measured in triplicates and a temperature ramp from 25-96°C. programmed. The fluorescence signal was acquired and the raw data wasanalyzed with the GraphPad Prism (GraphPad Software Inc.).

Stress Stability Study

The protein was analyzed over the course of two weeks and storage at 37°C. with respect to oligomerization by size-exclusion high-performanceliquid chromatography (SE-HPLC) and degradation by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Prior to thestudy the samples were concentrated to 1, 10 and 40 g/L and t0 timepoints were determined. The monomer content was quantified by separationof the samples on a Shodex KW-402.5-4F (Showa Denko) and evaluation ofthe resulting chromatograms. For the calculation of the relativepercentage of protein monomer the area of the monomeric peak was dividedby the total area of peaks that could not be attributed to the samplematrix. The protein degradation was assessed by SDS-PAGE analysis withAny kD Mini-Protean TGX gels (Bio-Rad Laboratories) and stained withCoomassie brilliant blue. The protein concentration was monitored at thedifferent time points by UV-Vis spectroscopy with an Infinity readerM200 Pro equipped with Nanoquant plate (Tecan Group Ltd.).

Example 3 Construction of scFv Constructs from a Marketed HumanizedMouse Monoclonal Antibody

To confirm the broad applicability of the proposed concept for thestabilization of antibody variable domains, two single-chain Fvconstructs were generated based on a marketed humanized mouse monoclonalantibody comprising a Vkappa light chain. For the first construct theoriginal variable domain sequences were used as published for the aminoacid sequence of the IgG, whereas for the second construct a modifiedsequence was used where the framework region IV of the variable lightchain domain was replaced by the respective sequence from a lambdagermline gene. In the scFv constructs the variable domains were linkedby a 20 amino acid flexible (Gly₄Ser)₄ linker (SEQ ID NO: 1), thusresulting in a configuration of NH₂-VL-linker-VH-COOH as used in modelconstructs scFv1 to scFv10 (see Table 2). The expression and refoldingof the scFv molecules were performed as described above, and therefolded proteins were purified by affinity chromatography over proteinL resin. Analysis of the purified proteins by SE-HPLC revealed markeddifferences in the producibility of the constructs that manifested in asignificantly improved monomer content of the construct containing thelambda framework IV in the VL (see FIG. 5). In addition the thermalunfolding analysis of the construct with the original sequence and themolecule containing the lambda framework IV in the VL resulted in amidpoint of unfolding of 69.9 and 71.2° C., respectively. Thisobservation is in line with the results described in Section [0082].

Example 4 Construction of scFv Constructs with a Shorter Linker Sequence

In order to analyze the impact of the linker sequence, alternativeconstructs were made in the same way as described above in Example 1using a shorter 15 amino acid (Gly₄Ser)₃ linker (SEQ ID NO: 34), Exceptfor the linker, the two constructs, scFv11 and scFv12, correspond toconstructs scFv1 and scFv5, respectively (see Table 2). Astress-stability study was performed as described above in Example 2. Asshown in Table 4, the construct scFv12 with the shorter linkercontaining the lambda framework IV in the VL showed an increasedstability relative to the construct with the consensus kappa light chainscFv11. The overall stability of the molecules was lower than thecorresponding constructs with the longer linker. It is well known thatscFv stability is increased by using 20mer or 25mer linkers instead of15mer linkers (see Wörn and Plückthun, J. Mol. Biol. 305 (2001)989-1010). Thus these findings are in line with results with 20merlinkers and confirm that the stability of a variable domain is improvedby introducing a lambda framework IV in the VL.

TABLE 1  List of protein sequences SEQ ID  NO: Type Sequence  1 LinkerGGGGSGGGGSGGGGSGGGGS  2 VL DIQMTQSPSSLSASVGDRVTITCQASQSISDWLAWYQQKPGKAPKLLIYGASRLASGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQGWSDSYVDNLFGQGTKVEIKR  3 VHEVQLVESGGGLVQPGGSLRLSCAVSGFSLSSGAMSWVRQAPGKGLEWIGVIISSGATYYASWAKGRFTISKDNSKNTVYLQMNSLRAEDTAVYYCARGGPDDSNSMGTFDPWGQGT LVTVSS  4 VLEIVMTQSPSTLSASVGDRVIITCQASQSISDWLAWYQQKPGKAPKLLIYGASRLASGVPSRFSGSGSGAEFTLTISSLQPD DFATYYCQQGWSDSYVDNLFGQGTKLTVLG 5 VH EVQLVESGGGLVQPGGSLRLSCTVSGFSLSSGAMSWVRQAPGKGLEWVGVIISSGATYYASWAKGRFTISKDTSKNTVYLQMNSLRAEDTAVYYCARGGPDDSNSMGTFDPWGQGT LVTVSS  6 VLEIVMTQSPSTLSASVGDRVIITCQASQSISDWLAWYQQKPGKAPKLLIYGASRLASGVPSRFSGSGSGAEFTLTISSLQPD DFATYYCQQGWSDSYVDNLFGQGTKVEIKR 7 VL DIQMTQSPSSLSASVGDRVTITCQASQSISDWLAWYQQKPGKAPKLLIYGASRLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGWSDSYVDNLFGQGTKLTVLG  8 VLDIQMTQSPSSLSASVGDRVTITCQASQSISDWLAWYQQKPGKAPKLLIYGASRLASGVPSRFSGSGSGTDFTLTISSLQPEDEATYYCQQGWSDSYVDNLFGQGTKLTVLG  9 VLDIQMTQSPSSLSASVGDRVTITCQASQSISDWLAWYQQKPGKAPKLLIYGASRLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGWSDSYVDNLFGGGTKLTVLG 10 VLDIQMTQSPSSLSASVGDRVTITCQASQSISDWLAWYQQKPGKAPKLLIYGASRLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGWSDSYVDNLFGQGTKVTVLG 11 VLDIQMTQSPSSLSASVGDRVTITCQASQSISDWLAWYQQKPGKAPKLLIYGASRLASGVPSRFSGSGSGTDFTLTISSLQPEDEATYYCQQGWSDSYVDNLFGGGTKLTVLG 12 VLDIQMTQSPSSLSASVGDRVTITCQASQSISDWLAWYQQKPGKAPKLLIYGASRLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGWSDSYVDNLFGTGTKVTVLG 13 VLDIQMTQSPSSLSASVGDRVTITCQASQSISDWLAWYQQKPGKAPKLLIYGASRLASGVPSRFSGSGSGTDFTLTISSLQPEDEATYYCQQGWSDSYVDNLFGTGTKVTVLG 14 VHEVQLVESGGGLVQPGGSLRLSCAASGFSLSSGAMSWVRQAPGKGLEWIGVIISSGATYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCARGGPDDSNSMGTFDPWGQGT LVTVSS 15 1.4gen FGQGTKLTVLGframework region IV 16 FR_IV Vλ FGTGTKVTVLG germ line 17 FR_IV VλFGGGTKLTVLG germ line 18 FR_IV Vλ FGGGTQLIILG germ line 19 FR_IV VλFGEGTELTVLG germ line 20 FR_IV Vλ FGSGTKVTVLG germ line 21 FR_IV VλFGGGTQLTVLG germ line 22 FR_IV Vλ FGGGTQLTALG germ line 23 Vk1DIQMTQSPSSLSASVGDRVTITC

WYQQKP consenus GKAPKLLIY

GVPSRFSGSGSGTDFTLTISSLQPE (rearranged) DFATYYC

FGQGTKVEIKR 24 Vk2 DIVMTQSPLSLPVTPGEPASISC

W consensus YLQKPGQSPQLLIYL

GVPDRFSGSGSGTDFTLKI (rearranged) SRVEAEDVGVYYC

FGQGTKLEIKR 25 Vk3 EIVLTQSPGTLSLSPGERATLSC

WYQQK consensus PGQAPRLLIY

GIPDRFSGSGSGTDFTLTISRLEP (rearranged) EDFAVYYC

FGQGTKVEIKR 26 Vk4 DIVMTQSPDSLAVSLGERATINC

consensus WYQQKPGQPPKLLIY

GVPDRFSGSGSGTDFTL (rearranged) TISSLQAEDVAVYYC

FGQGTKVEIKR 27 VH1A QVQLVQSGAEVKKPGSSVKVSCKASG

WVRQ consensus APGQGLEWMG

RVTITADESTSTA (rearranged) YMELSSLRSEDTAVYYCAR

WGQGTLVTVS S 28 VH1B QVQLVQSGAEVKKPGASVKVSCKASG

WVR consensus QAPGQGLEWMG

RVTMTRDTSI (rearranged)  STAYMELSSLRSDDTAVYYCAR

WGQGTLVT VSS 29 VH2 QVTLKESGPALVKPTQTLTLTCTFSG

WIR consensus QPPGKALEWLA

RLTISKDTSKNQV (rearranged) VLTMTNMDPVDTATYYCAR

VGQGTLVTVSS 30 VH3 EVQLVESGGGLVQPGGSLRLSCAASG

WVR consensus QAPGKGLEWVS

RFTISRDNSKN (rearranged) TLYQMNSLRAEDTAVYYCAR

WGQGTLVTVSS 31 VH4 QVQLQESGPGLVKPSETLSLTCTVSG

WIRQP consensus PGKGLEWIG

RVTISVDTSKNQFSL (rearranged) KLSSVTAADTAVYYCAR

WGQGTLVTVSS 32 VH5 EVQLVQSGAEVKKPGESLKISCKGSG

WVRQ consensus MPGKGLEWMG

QVTISADKSISTA (rearranged) YLQWSSLKASDTAMYYCAR

WGQGTLVTV SS 33 VH6 QVQLQQSGPGLVKPSQTLSLTCAISG

WI consensus RQSPSRGLEWLG

RITINPDTS (rearranged) KNQFSLQLNSVTPEDTAVYYCAR

VGQGTLVTV SS 42 Linker GGGGSGGGGSGGGGS (in SEQ ID NOs: 23 to 33, the CDRregions are indicated in bold and italic letters)

TABLE 2 Combinations of variable domains for the different scFvconstructs scFv Variable Variable construct domain 1 Linker domain 2scFv1 SEQ ID 2 SEQ ID 1 SEQ ID 3 scFv2 SEQ ID 4 SEQ ID 1 SEQ ID 5 scFv3SEQ ID 6 SEQ ID 1 SEQ ID 3 scFv4 SEQ ID 11 SEQ ID 1 SEQ ID 14 scFv5 SEQID 9 SEQ ID 1 SEQ ID 14 scFv6 SEQ ID 8 SEQ ID 1 SEQ ID 14 scFv7 SEQ ID 7SEQ ID 1 SEQ ID 14 scFv8 SEQ ID 13 SEQ ID 1 SEQ ID 14 scFv9 SEQ ID 12SEQ ID 1 SEQ ID 14 scFv10 SEQ ID 10 SEQ ID 1 SEQ ID 14 scFv11 SEQ ID 2SEQ ID 42 SEQ ID 3 scFv12 SEQ ID 9 SEQ ID 42 SEQ ID 14

TABLE 3 The midpoint of transition for the thermal unfolding wasdetermined for all constructs by differential scanning fluorimetryConstruct ID Tm scFv 1 70.19 ± 0.22 scFv 2 66.82 ± 0.37 scFv 3 65.44 ±0.15 scFv 4 74.31 ± 0.04 scFv 5 70.86 ± 0.16 scFv 6 75.19 ± 0.13 scFv 768.42 ± 0.05 scFv 8 75.45 ± 0.36 scFv 9 71.15 ± 0.27 scFv 10 71.25 ±0.29 scFv 11 70.34 ± 0.20 scFv 12 70.18 ± 0.03

TABLE 4 Monomer loss during storage SEQ ID 1 g/L at 37° C. 10 g/L at 37°C. 40 g/L at 37° C. scFv 1 −16.8% −44.3% −34.7% scFv 2 −1.9% −20.1%−39.6% scFv 3 −23.0% −64.7% −81.0% scFv 4 −0.9% −9.2% −14.0% scFv 5−1.6% −11.4% −17.0% scFv 6 −0.8% −4.0% −6.5% scFv 7 −0.6% −13.1% −25.4%scFv 8 0.1% −0.2% −3.1% scFv 9 −1.3% −10.1% −22.9% scFv 10 −0.5% −14.4%−26.7% scFv 11 −56.3% −77.7% −80.2% scFv 12 −5.7% −41.3% −65.9% NOTE:entries in italics are from scFv constructs with Vκ-type framework IVregions

TABLE 5 List of clones listed in Borras et al. (loc. cit.) and sequencevariations All clones listed below have the sequence of FW1.4gen (SEQ IDNO: 4), but differ in the positions indicated below in bold letters(numbering of positions according to Borras et al. (loc. cit.)) VLposition 15 22 40 49 58 69 70 72 74 77 79 81 FW1.4gen V T P Y V A E T TS Q D (SEQ ID NO: 4) 375-FW1.4opt V T P Y V T Q T T S Q D 435-FW1.4opt VK P Y V A E T T S Q D 509-FW1.4opt V T P Y V T E T T S Q D 511-FW1.4optV T P Y V T E T T S Q D 534-FW1.4opt V T P Y V T E T T S Q D567-FW1.4opt V T P Y V T Q T T S Q D 578-FW1.4opt V T P Y V T Q T T S QD  1-FW1.4opt V T P Y V T E T T S Q D  8-FW1.4opt V T P Y V T D T A S QD  15-FW1.4opt V T P Y V T E T T S Q D  19-FW1.4opt V T P Y V T Q T T SQ D  34-FW1.4opt L T S Y V A E S T S Q D  35-FW1.4opt V T P Y V T E T TS Q D  42-FW1.4opt V T P Y V T E T T S Q D  43-FW1.4opt V K P Y F A E TT G E A

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

To the extent possible under the respective patent law, all patents,applications, publications, test methods, literature, and othermaterials cited herein are hereby incorporated by reference.

We claim:
 1. An antibody VL domain comprising (i) human Vκ frameworkregions I to III; (ii) CDR domains CDR1, CDR2 and CDR3; and (iii) aframework region IV, which is selected from a. a human Vλ germ linesequence for framework region IV, particularly a Vλ germ line sequenceselected from the list of: SEQ ID NO. 16 to 22; b. a Vλ-based sequence,which is (bi) a consensus Vλ sequence from human Vλ germ line sequencesfor framework region IV, particularly SEQ ID NO. 17; or (bii) aconsensus Vλ sequence from rearranged human Vλ sequences for frameworkregion IV, particularly a Vλ consensus sequence selected from the listof: SEQ ID NO. 16 and 17; and c. a Vλ-based sequence, which has one ortwo mutations, particularly one mutation, compared to the closest humanVλ germ line sequence for framework region IV; provided that if, in thecase of b. or c., framework region IV has the sequence FGQGTKLTVLG (SEQID No. 15) (w) said human Vκ framework regions I to III are differentfrom the framework regions as found in the list of clones: FW1.4gen (SEQID NO: 4), 375-FW1.4opt, 435-FW1.4opt, 509-FW1.4opt, 511-FW1.4opt,534-FW1.4opt, 567-FW1.4opt, 578-FW1.4opt, 1-FW1.4opt, 8-FW1.4opt,15-FW1.4opt, 19-FW1.4opt, 34-FW1.4opt, 35-FW1.4opt, 42-FW1.4opt, and43-FW1.4opt; (x) said human Vκ framework regions I to III are differentfrom a sequence obtainable by permutation from the sequences of theframework regions as found in the list of clones: FW1.4gen (SEQ ID NO:4), 375-FW1.4opt, 435-FW1.4opt, 509-FW1.4opt, 511-FW1.4opt,534-FW1.4opt, 567-FW1.4opt, 578-FW1.4opt, 1-FW1.4opt, 8-FW1.4opt,15-FW1.4opt, 19-FW1.4opt, 34-FW1.4opt, 35-FW1.4opt, 42-FW1.4opt, and43-FW1.4opt; (y) said human Vκ framework regions I to III are differentfrom a sequence obtainable by mutation of the sequence FW1.4gen (SEQ IDNO: 4) at one or more of positions 15, 22, 48, 57, 74, 87, 88, 90, 92,95, 97 and 99; or (z) said human Vκ framework regions I to III comprisenot more than five mutations compared to the respective regions in thehuman Vκ sequence with SEQ ID No: 8, particularly less than five, lessthan four, less than three, particularly only one or no mutationcompared to the human Vκ sequence with SEQ ID No:
 8. 2. The antibody VLdomain of claim 1, wherein the amino acid residue in position AHo101 inframework region III is an amino acid residue present at that positionin a human Vλ consensus sequence, particularly wherein said amino acidresidue is different from phenylalanine, more particularly wherein saidamino acid residue is glutamic acid.
 3. The antibody VL domain of claim1, wherein said Vκ framework regions I to III are from the Vκ1 family.4. The antibody VL domain of claim 3, wherein said Vκ framework regionsI to III are the framework regions present in a sequence selected fromSEQ ID NO: 2 and SEQ ID NO: 8, particularly SEQ ID NO:
 8. 5. Theantibody VL domain of claim 1, wherein said CDR domains CDR1, CDR2 andCDR3 are independently selected from (i) CDR domains CDR1, CDR2 and CDR3from a parental non-human antibody with specificity for an antigen ofinterest, particularly from a parental rabbit antibody or from aparental rodent antibody, particularly a parental mouse or rat antibody;(ii) CDR domains CDR1, CDR2 and CDR3 from a parental human or humanizedantibody comprising a Vκ domain, particularly from an antibody approvedfor therapy or otherwise being commercialized; (iii) CDR domains CDR1,CDR2 and CDR3 derived from the CDR domains according to (i) or (ii),particularly CDR domains obtained by optimizing one or more of the CDRdomains according to (i) or (ii); and (iv) a CDR domain to be replacedby one or more CDR domains according to (i), (ii) and/or (iii).
 6. Theantibody VL domain of claim 1, wherein the framework regions I to IV area combination of framework regions as found in a sequence selected from:SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO:13.
 7. An isolated antibody or functional fragment thereof comprising anantibody VL domain according to claim
 1. 8. The isolated antibody orfunctional fragment thereof of claim 7, comprising a VH domain belongingto a VH domain subfamily selected from VH1A, VH1B, VH2, VH3, VH4, VHS,and VH6, particularly to a VH domain subfamily VH3 or VH4, particularlyto the VH domain subfamily VH3.
 9. The isolated antibody or functionalfragment thereof of claim 7, which is selected from the group consistingof: an IgG antibody, a Fab fragment, an scFv fragment, a single-chaindiabody (scDb), a tandem scDb (Tandab), a linear dimeric scDb (LD-scDb),a circular dimeric scDb (CD-scDb), a bispecific T-cell engager (BiTE;tandem di-scFv), a tandem tri-scFv, a tri(a)body, bispecific Fab2,di-miniantibody, tetrabody, scFv-Fc-scFv fusion, di-diabody, DVD-Ig,IgG-scFab, scFab-dsscFv, Fv2-Fc, IgG-scFv fusions, such as bsAb (scFvlinked to C-terminus of light chain), Bs1Ab (scFv linked to N-terminusof light chain), Bs2Ab (scFv linked to N-terminus of heavy chain), Bs3Ab(scFv linked to C-terminus of heavy chain), Ts1Ab (scFv linked toN-terminus of both heavy chain and light chain), Ts2Ab (dsscFv linked toC-terminus of heavy chain), and Knob-into-Holes (KiHs) (bispecific IgGsprepared by the KiH technology) and DuoBodies (bispecific IgGs preparedby the Duobody technology)
 10. A pharmaceutical composition comprisingthe isolated antibody or functional fragment thereof of claim 7, andoptionally a pharmaceutically acceptable carrier and/or excipient.
 11. Anucleic acid sequence or a collection of nucleic acid sequences encodingthe antibody VL domain of claim 1, or an isolated antibody or functionalfragment comprising an antibody VL domain according to claim
 1. 12. Avector or a collection of vectors comprising the nucleic acid sequenceor a collection of nucleic acid sequences of claim
 11. 13. A host cell,particularly an expression host cell, comprising the nucleic acidsequence or the collection of nucleic acid sequences of claim 11, or avector or collection of vectors comprising the nucleic acid sequence ora collection of nucleic acid sequences of claim
 11. 14. A method forproducing the antibody VL domain of claim 1, or an isolated antibody orfunctional fragment comprising an antibody VL domain according to claim1 comprising the step of expressing the nucleic acid sequence or thecollection of nucleic acid sequences encoding said antibody VL domainaccording to claim 1 or said isolated antibody or functional fragmentcomprising an antibody VL domain according to claim 1 or a vector orcollection of vectors comprising said nucleic acid sequence or saidcollection of nucleic acid sequences optionally in a host cell,particularly an expression host cell.
 15. A method for generating ahumanized rabbit or rodent antibody comprising the steps of: a.immunization of rabbits or rodents with an antigen of interest; b.isolating at least one antibody of interest; and c. cloning of the VLCDR regions of said at least one antibody of interest into a nucleicacid sequence encoding an antibody VL domain of claim
 1. 16. The methodof claim 15, further comprising one or more of the steps of: aa. clonalisolation of affinity matured memory B-cells that interact with theantigen of interest, particularly by using fluorescence activatedcell-sorting; ab. cultivation of single B cells in a co-cultivationsystem that does not require immortalization of single B cell clones;ac. screening of B cell culture supernatants in a cell-based ELISA toidentify at least one antibody binding to the antigen of interest;and/or ad. cloning of the VH CDR regions of at least one antibody into anucleic acid sequence encoding a human antibody VH domain.
 17. A methodfor generating a nucleic acid sequence encoding an antibody VL domain ofclaim 1, or one or more nucleic acid sequences encoding the isolatedantibody or functional fragment comprising an antibody VL domainaccording to claim 1, comprising combining in one or more steps nucleicacid sequences encoding (i) human Vκ framework regions I to III; (ii)CDR domains CDR1, CDR2 and CDR3, and (iii) a framework region IV, whichis selected from a. a human Vλ germ line sequence for framework regionIV, particularly a Vλ germ line sequence selected from the list of: SEQID NO. 16 to 22; b. a Vλ-based sequence, which is (bi) a consensus Vλsequence from human Vλ germ line sequences for framework region IV,particularly SEQ ID NO. 17; or (bii) a consensus Vλ sequence fromrearranged human Vλ sequences for framework region IV, particularly a Vλconsensus sequence selected from the list of: SEQ ID NO. 16 and 17; andc. a Vλ-based sequence, which has one or two mutations, particularly onemutation, compared to the closest human Vκ germ line sequence forframework region IV; particularly using one of the following methods: i.replacing in a nucleic acid construct, particularly in a recombinantvector, comprising a nucleic acid sequence encoding a human or humanizedVκ domain the Vκ framework region IV by a framework region IV, which isselected from a. a human Vλ germ line sequence for framework region IV,particularly a Vλ germ line sequence selected from the list of: SEQ IDNO. 16 to 22; b. a Vλ-based sequence, which is (bi) a consensus Vλsequence from human Vλ germ line sequences for framework region IV,particularly SEQ ID NO. 17; or (bii) a consensus Vλ sequence fromrearranged human Vλ sequences for framework region IV, particularly a Vλconsensus sequence selected from the list of: SEQ ID NO. 16 and 17; andc. a Vλ-based sequence, which has one or two mutations, particularly onemutation, compared to the closest human Vλ germ line sequence forframework region IV; ii. inserting in one or more steps into a nucleicacid construct, particularly into a recombinant vector, comprising anucleic acid sequence encoding a framework region IV one or more nucleicacid sequences encoding (i) human Vκ framework regions I to III; and(ii) CDR domains CDR1, CDR2 and CDR3, wherein said framework region IVis selected from a. a human Vλ germ line sequence for framework regionIV, particularly a Vλ germ line sequence selected from the list of: SEQID NO. 16 to 22; b. a Vλ-based sequence, which is (bi) a consensus Vλsequence from human Vλ germ line sequences for framework region IV,particularly SEQ ID NO. 17; or (bii) a consensus Vλ sequence fromrearranged human Vλ sequences for framework region IV, particularly a Vλconsensus sequence selected from the list of: SEQ ID NO. 16 and 17; andc. a Vλ-based sequence, which has one or two mutations, particularly onemutation, compared to the closest human Vλ germ line sequence forframework region IV; iii. mutating in a nucleic acid sequence encoding ahuman or humanized Vκ domain the nucleic acid sequence encodingframework region IV to generate a framework region IV, which is selectedfrom a. a human Vλ germ line sequence for framework region IV,particularly a Vλ germ line sequence selected from the list of: SEQ IDNO. 16 to 22; b. a Vλ-based sequence, which is (bi) a consensus Vλsequence from human Vλ germ line sequences for framework region IV,particularly SEQ ID NO. 17; or (bii) a consensus Vλ sequence fromrearranged human Vλ sequences for framework region IV, particularly a Vλconsensus sequence selected from the list of: SEQ ID NO. 16 and 17; andc. a Vλ-based sequence, which has one or two mutations, particularly onemutation, compared to the closest human Vλ germ line sequence forframework region IV; or iv. replacing in one or more steps in a nucleicacid construct, particularly in a recombinant vector, comprising anucleic acid sequence encoding a light chain domain comprising human Vκframework regions I to III, CDR domains CDR1, CDR2 and CDR3, and aframework region IV, one or more of the nucleic acid sequences encodingsaid CDR domains by nucleic acid sequence(s) encoding the correspondingCDR domain(s) from an antibody of interest, wherein said frameworkregion IV is selected from a. a human Vλ germ line sequence forframework region IV, particularly a Vλ germ line sequence selected fromthe list of: SEQ ID NO. 16 to 22; b. a Vλ-based sequence, which is (bi)a consensus Vλ sequence from human Vλ germ line sequences for frameworkregion IV, particularly SEQ ID NO. 17; or (bii) a consensus Vλ sequencefrom rearranged human Vλ sequences for framework region IV, particularlya Vλ consensus sequence selected from the list of: SEQ ID NO. 16 and 17;and c. a Vλ-based sequence, which has one or two mutations, particularlyone mutation, compared to the closest human Vλ germ line sequence forframework region IV.
 18. A nucleic acid sequence or a collection ofnucleic acid sequences obtainable by the method of claim 17.